This invention relates to recombinant protein production and, more particularly, to a high pressure, infrared spectroscopic technique for monitoring such recombinant protein production.
The production of many prokaryotic and eukaryotic proteins in Escherichia coli has been made possible through the use of recombinant DNA technology. High-level expression of these proteins has been achieved by cloning their coding sequences into multicopy plasmids downstream from strong promoters and ribosome binding sites to create recombinant expression plasmids. Using this approach, several investigators have reported that products of cloned genes can accumulate in certain cases up to 50% of total E. coli cell protein.
It has been observed that high level expression of these cloned genes resulted in the formation of insoluble proteinaceous aggregates. Also, many of the proteins may be found in the soluble fractions. It has, for instance, been found recently that the rapid degradation of human proinsulin synthesized in E. coli by recombinant DNA techniques can be prevented by inserting a DNA sequence encoding a short homooligopeptide, such a (Ala).sub.6, (Asn).sub.6, (Cys).sub.7, (Gln).sub.7, (His).sub.6, (Ser).sub.6 and (Thr).sub.6, at the 5' end of the proinsulin gene. The expressed polypeptide is then accumulated as cytoplasmic inclusion bodies, with the yield of proinsulin ranging between 6% and 26% of the total bacterial protein.
For large scale production of proinsulin and other proteins, an efficient method for direct determination of maximum gene expression and detection of any biological contamination would be important economically. However, the established procedures of radioimmunoassay and SDS-polyacrylamide gel electrophoresis are time-consuming, and therefore totally inappropriate as a monitoring process for commercial production.